Peripheral T cells can be divided into two distinct subpopulations based on their mutually exclusive expression of the cell surface glycoproteins, CD4 and CD8. The CD4+CD8- cells are primarily helper T cells and express TCRs restricted by class II MHC proteins; CD4-CD8+ cells are predominately cytotoxic cells and express TCRs restricted by class I MHC molecules. CD4 and CD8 bind directly to conserved sites on MHC class II and class I molecules, respectively; this binding most likely functions to increase the avidity of T cell:target cell interaction, not only in an immune response, but also during T cell maturation in the thymus. The primary objective of the proposed research is to further investigate the regulation and function of CD8 and CD4 during thymocyte differentiation. Our main approach involves the manipulation of CD8alpha and CD8beta expression in transgenic mice; four different experimental strategies are described to test models of CD8 and CD4 regulation and function. (1) Identification of T cell- specific regulatory sequences. The cis-acting elements that control the T cell subclass-specific expression of the mouse and human CD8 gene loci will be identified. For this purpose, the linkage arrangement of the human CD8alpha and beta genes will first be established by the isolation and mapping of YAC clones. Potential T cell-specific regulatory elements will be located in both the human and mouse CD8 loci by mapping DNAse I hypersensitive sites. These sites will be tested for thymocyte and/or CD8+ T cell-specific enhancer activity in both cultured cells and transgenic mice. (2) Deregulation of CD8alpha and Cd8beta in transgenic mice. Transgenic mice carrying the mouse CD8alpha and CD8beta genes under the control of pan T cell-specific enhancers and promoters will be generated and analyzed by FACS, MLR and CTL assays and by crosses with transgenic mice expressing a class I restricted TCR. (3) Generation of null mutations in CD8alpha and CD8beta in transgenic mice. Using gene targeting in embryonic stem (ES) cells, we plan to generate CD8alpha and CD8beta deficient mice in order to further explore the function of CD8 in T cell development. (4) Generation of HLA class I transgenic mice. Human CD8 transgenic mice will be mated to HLA B7 or A2 transgenic mice to determine if the selection of an HLA class I molecule as a restriction element is dependent on its interaction with human CD8. If selection is found to be dependent on human CD8, then this transgenic mouse system can be used for structure-function assays to identify the regions of the CD8 protein that are critical for T cell selection in the thymus.